KR20130099927A - Simplified basic media for human pluripotent cell culture - Google Patents

Abstract

A fully defined medium that supports multipotent cell survival, proliferation, cloning and induction, and methods and compositions comprising the medium are described. Also described is a method of inducing iPS cells from an adult individual under defined heterogeneous material free conditions.

Description

Simplified basal medium for culturing human multipotent cells {SIMPLIFIED BASIC MEDIA FOR HUMAN PLURIPOTENT CELL CULTURE}

Cross-reference to related application

Not Applicable

Reference to federal funding research or development

The present invention was made with government support under ES017166 granted by the National Institutes of Health. The United States government has certain rights in the invention.

Multipotent cells, such as embryonic stem (ES) cells and induced pluripotent stem cell (iPS) cells, have the potential to differentiate into all three primary germ cells (Thomson, et al. , Science 282, 1145-1147 (1998). The surprising developmental potential of multipotent cells has proven useful for basic research and clinical applications. Numerous basic methods for culturing human multipotent cells, such as growth medium, plate coating and other conditions, have been developed and improved (Ludwig et al. , Nat. Biotechnol 24, 185-187 (2006); Ludwig et al. , Nat. Methods 3, 637-646 (2006). For example, human ES cells were initially cultured in fetal bovine serum (FBS) containing medium on murine embryonic fibroblast (MEF) feeder cells, but now regulatory protein matrices as well as fully defined medium are used. Possible (Ludwig et al. , Nat. Biotechnol 24, 185-187 (2006)).

Over the past decade, pluripotent cell cultures have evolved significantly. Several proliferation media have been developed that provide basic nutrients and growth factors for the survival and proliferation of multipotent cells and directly determine how cells grow and differentiate. TeSR ^™ was one of the first defined media to support pluripotent cell maintenance in undifferentiated state in the absence of feeder cells or conditioned medium through multiple culture passages (Ludwig et al. , Nat. Methods 3, 637-). 646 (2006); US Pat. No. 7,449,334, each of which is incorporated herein by reference in its entirety). TeSR ^™ itself contains 18 components in addition to basal medium DMEM / F12, which contains 52 components (Table 1).

The variety of different growth media available for pluripotent cell culture has contributed to the inconsistency of the study results. Currently used media for inducing and growing pluripotent cells, including fully defined media, contain components that can affect pluripotent cells in a variety of ways. Prior to the present invention described herein, it is not known that each media component, alone or in combination with other components, affects various multipotent cell functions such as viability, multipotency or differentiation in cell culture.

For example, albumin, the most abundant protein component present in most media, is a lipid carrier and thus can affect differentiation or maintenance of multidifferentiation through bound lipids. The properties of albumin and its associated lipids determine whether it can be used for human multipotent cell culture. However, albumin properties, even when made from recombinant genetic material, vary greatly depending on their source, contributing to the variation between experiments performed under other equivalent conditions. In addition, cloned human serum albumin is available, but due to its relatively high price it is rarely used in conventional experiments.

Efforts to remove albumin from the media have proven unsuccessful. Omission of albumin or any other growth factor present in TeSR resulted in a significant decrease in human ESC culture efficiency, such as a decrease in cell viability, growth and multipotency (Ludwig et al. , Nat. Biotechnol 24, 185-187 ( 2006).

In order to fully exploit the potential of multipotent cells for drug development, testing and transplantation therapy, induction and proliferation of these cells under fully defined conditions, ideally xeno-free conditions, is desirable. Thus, there is a need in the art to meet the need for a medium that does not contain components that introduce inconsistencies to maintain control over multipotent cell culture conditions. In particular, there is a need in the art for multipotent cell culture media containing only components that support multipotent cell function that are important for specific culture purposes.

The present invention generally relates to media, compositions and methods for inducing and culturing multipotent cells, and more particularly to fully defined media for multipotent cells.

In a first aspect, the present invention is summarized as an albumin free medium that supports the viability, growth and multipotency of multipotent cells.

In some embodiments of the first aspect, the medium comprises selenium.

In some embodiments of the first aspect, the medium comprises NODAL.

In some embodiments of the first aspect, the medium comprises transferrin.

In some embodiments of the first aspect, the medium comprises transforming growth factor beta (TGF-β).

In some embodiments of the first aspect, the medium comprises only water, salts, amino acids, vitamins, carbon sources, insulin and fibroblast growth factor (FGF) in amounts sufficient to support multipotent stem cell viability, respectively.

In some embodiments of the first aspect, the medium only contains water, salts, amino acids, vitamins, carbon sources, insulin, FGF, selenium, transferrin, and one of TGF-β and NODAL, respectively, to support multipotent stem cell proliferation. Included in quantity.

In some embodiments of the first aspect, the medium supports viability after passage, freezing, proliferation, multipotency, induction and cloning of multipotent cells.

In some embodiments of the first aspect, the medium is heterogeneous free.

In a second aspect, the invention is summarized as a method of culturing multipotent stem cells in a defined medium. In some embodiments of the second aspect, the medium used to culture the multipotent cells comprises only water, salts, amino acids, vitamins, carbon sources, insulin and FGF in amounts sufficient to support multipotent cell viability, respectively. In some embodiments of the second aspect, the medium used to culture the pluripotent cells is only water, salt, amino acid, vitamin, carbon source, insulin, FGF, selenium, transferrin, and one of TGF-β and NODAL, respectively. And in an amount sufficient to support pluripotent stem cell proliferation. In some embodiments of the second aspect, the medium comprises regulatory factors that support prolonged growth, multipotency, cloning, freezing or induction of multipotent cells. In some embodiments of the second aspect, the medium used to culture the multipotent cells is free of heterologous material.

In a third aspect, the invention relates to an in vitro cell culture composition of multipotent cells in a medium substantially free of β-mercaptoethanol and albumin. In some embodiments of the third aspect, the culture composition is fibroblast It does not include feeder cells, conditioned media, and heterologous contamination.

In a fourth aspect, the invention is summarized as a method of inducing iPS cells from an adult individual under fully defined conditions. The method comprises the steps of culturing somatic cells from an adult individual in a medium containing water, salts, amino acids, vitamins, carbon sources, insulin and FGF in an amount sufficient to maintain viability, and for example cells under defined conditions to induce iPS cells. Reprogramming.

In some embodiments of the fourth aspect, the medium comprises TGF-β in some or all of the reprogramming process.

In some embodiments of the fourth aspect, the medium comprises butyrate.

In some embodiments of the fourth aspect, the medium comprises hydrocortisone.

In some embodiments of the fourth aspect, the medium is heterogeneous free.

In a fifth aspect, the invention is summarized as a method of cloning multipotent stem cells in albumin free medium. The method includes plating the multipotent stem cells at the cloning density in albumin-free medium that supports multipotent stem cell cloning.

In some embodiments of the fifth aspect, the medium comprises a ROCK inhibitor.

In some embodiments of the fifth aspect, the medium comprises blevistatin.

In some embodiments of the fifth aspect, the medium only contains water, salts, amino acids, vitamins, carbon sources, insulin, FGF, selenium, transferrin, and one of TGF-β and NODAL, respectively, to support multipotent stem cell cloning. Included in quantity.

In a sixth aspect, the invention is summarized as a method of cryopreserve multipotent stem cells in albumin free medium. The method includes freezing multipotent stem cells in albumin free medium.

In some embodiments of the sixth aspect, the medium comprises only water, salts, amino acids, vitamins, carbon sources, insulin, FGF, selenium, transferrin; One of TGF-β and NODAL, and dimethyl sulfoxide (DMSO).

In a seventh aspect, the present invention is summarized as iPS cells induced under albumin condition. IPS cells induced in the absence of albumin do not contain endogenous albumin contamination.

The methods and compositions described herein are useful for a variety of uses for inducing, culturing and using multipotent cells. It is an object of the present invention to define short and long term culture conditions for multipotent cells defined as factors supporting the intended culture objectives.

Another object of the present invention is to provide culture conditions for multipotent cells that maximize the proportion of cultured cells in undifferentiated state.

Another object of the present invention is to provide a medium that can be used as a platform for observing how various conditions affect pluripotent cells and comparing them to previously reported experiments in various medium environments.

These and other features, objects, and advantages of the present invention will be better understood from the following detailed description. In the detailed description, reference is made to the accompanying drawings, which form a part hereof, wherein embodiments of the invention are illustrated without limitation. The description of the preferred embodiments is not intended to limit the invention and is intended to cover all modifications, equivalents, and alternatives. Accordingly, reference should be made to the claims cited herein to interpret the scope of the invention.

The present invention will be better understood upon consideration of the following detailed description, and features, aspects, and advantages other than those described above will become apparent. Such details refer to the following drawings.
1A-1E illustrate media elements for human ES cell survival and self-renewal in culture. 1A illustrates the 24-hour survival index for individualized cells plated in various media. Medium abbreviations are as listed in Table 1. The presence of insulin and fibroblast growth factor (IF), bovine serum albumin (BSA), beta-mercaptoethanol (BME) is indicated by "+" and the absence is indicated by "-". 1B illustrates 24 and 96 hour survival indices for individualized cells plated in various media. Addition of insulin and fibroblast growth factor (FGF) is indicated by "+" and removal is indicated by "-". 1C illustrates the 24-hour or 129-hour survival index for individualized cells cultured in vitamin C containing TeSR ^™ medium, vitamin C free TeSR ^™ medium (TeSR ^™ -LAA), or DF5 medium. 1D illustrates cell proliferation after each three passages in DF5, selenium-added DF5 (DF5 + selenium), DF12, or selenium-removed DF12 (DF12-selenium). 1E illustrates comparative analysis of 12 different basal media.
2A-2F illustrate optimization of human ES cells and iPS cell culture conditions by DF5S. FIG. 2A shows low density (˜1,500 cells / cm ² ) seeded in DF5S (bottom) or TeSR ^™ (top) and different O ₂ and CO ₂ concentrations (O 15 C 5: 15% O ₂ and 5% CO ₂ ; O 15 C 10: 15). % 0 ₂ and 10% C0 ₂ ; O5C10: survival index for individualized cells cultured in 5% O ₂ and 10% CO ₂ ). Cell viability was examined at 24 and 124 hours. FIG. 2B shows the cloning efficiency of H1 cells cultured in various media with or without small molecule HA100 (+ HA100) (CM100: Conditional medium with 100 ng / ml FGF). 2C shows the cloning efficiency of iPS cells and H1 cells derived from foreskin fibroblasts in various media. 2D shows the cloning efficiency of iPS cells derived from foreskin fibroblasts in various media. DF5S trFe represents DF5S medium to which holotransferrin was added. 2E shows the cloning efficiency of H1 cells cultured in various media in the presence of HA100 (10 μM, 24 hours), blevistatin (10 μM, 4 hours), or Y27632 (10 μM, 24 hours) without these factors. An example is compared with an efficiency (control). * Indicates p < 0.05. FIG. 2F illustrates the cloning efficiency of H1 cells in conditioned medium (CM), under ROCK inhibitor (HA100) containing CM, under ROCK inhibitor containing TeSR, and under ROCK inhibitor containing E8 under normal oxygen (dark gray bar) or low oxygen (light gray bar) conditions. do. Error bars represent standard error of the mean; * Indicates p < 0.05.
3A and 3B show DMEM supplemented with insulin, transferrin, selenium, L-ascorbic acid, FGF2, and TGF-β or NODAL (referred to herein as "E8 (TGF-β)" and "E8 (NODAL)", respectively). Multipotent cell growth and gene expression at / F12 are illustrated. 3A illustrates the proliferation fold of H1 ES cells (top) and iPS cells (bottom) maintained in TeSR ^™ (dark gray line) or E8 (TGF-β (light gray line). FIG. 3B shows E8 (TGF-β) Total gene expression of H1 ES cells cultured in H1 ES cells and H1 ES cells cultured in TeSR ^™ is illustrated RNA of H1 cells maintained for three passages in TeSR or E8 (TGFβ) medium is analyzed by RNA-using Illumina Genome Analyzer GAIIX. Analysis by seq (total gene expression correlation R = 0.954 (Spearman Correlation)).
4A-4F illustrate iPS cell induction under defined conditions. 4A illustrates the proliferation of foreskin fibroblasts in DF5SFe basal medium supplemented with various fibroblast growth factor (FGF) compared to proliferation in FBS containing medium. 4B shows fibroblast growth in various media supplemented with hydrocortisone. 4C depicts expression of the multipotent markers OCT4 (left) and SSEA4 (right). 4D illustrates expression of selected genes by foreskin fibroblasts, hES cells, iPS cells induced on feeder cells (iPS cells (feeders)), and iPS cells induced in E8 medium (iPS cells (E8)). All cells were maintained in E8 (TGFβ) medium before RNA analysis, except fibroblasts were maintained in E8 including hydrocortisone. 4E illustrates total gene expression of human ES and iPS cells induced in E8 (TGFβ) medium (R = 0.95). 4F illustrates total gene expression of iPS cells induced on MEF and iPS cells induced in E8 (TGFβ) medium.
5A-5C illustrate media improvement for iPS cell induction. FIG. 5A shows foreskin (dark gray bar) and PRPF8-2 adult fibroblasts (light gray bar) in DF5SFe medium supplemented with TGF-β, hydrocortisone, TGF-β and hydrocortisone, or TGF-β and hydrocortisone without FGF Show proliferation. 5B illustrates the effect of TGF-β and butyrate on reprogramming of foreskin fibroblasts. Four to five weeks after initial reprogramming transfection, colony counts for transformed cells and true iPS cells were scored and the ratio of iPS colonies to non-iPS cell colonies was calculated.
6A and 6B illustrate the induction of iPS cells from adult fibroblasts under fully defined conditions without secondary passage. 6A illustrates an example of a reprogramming protocol. FIG. 6B shows multiple minutes measured by flow cytometry of iPSC cell lines maintained for 20 passages in DMEM / F12 (“E8”) supplemented with insulin, transferrin, selenium, L-ascorbic acid, FGF2, and TGF-β or NODAL. Expression of the chemical markers OCT4 and SSEA4 is illustrated. Shaded peak: stained with antibodies specific for OCT4 (left) and SSEA4 (right) (left); Non-shaded peak: mouse IgG control antibody.
7A-7C illustrate the reprogramming efficiency of human fibroblasts in various media. FIG. 7A illustrates the number of iPS cell colonies per 80,000 fibroblasts applied to reprogramming with mouse fibroblast feeder cells (MEF) or in E8 based media. To improve the efficiency, 100 μM sodium butyrate was added to both conditions. FIG. 7B illustrates the number of iPS cell colonies per 80,000 fibroblasts applied for reprogramming in TeSR ^™ or in E8 based media. 7C illustrates the effect of TGF-β and butyrate exposure time on reprogramming efficiency of foreskin fibroblasts under fully defined conditions. Fibroblasts were reprogrammed in DMEM / F12 (TGF-β free E8) or E8 supplemented with insulin, transferrin, selenium, L-ascorbic acid and FGF2 with or without 100 μM butyrate. Reprogramming efficiencies for all conditions were analyzed 30 days after reprogramming. * Indicates p &lt; 0.05.
While the invention is susceptible to various modifications and alternative forms, exemplary embodiments thereof have been described by way of example with reference to the drawings and are described in detail herein. However, the description of the exemplary embodiments is not intended to limit the invention to the particular forms disclosed, and on the contrary, the intention is that all modifications belong to the spirit and scope of the invention as defined by the appended claims. , Equivalents and alternatives.

The present invention relates to the inventors' observation that certain media components that were considered essential for the culturing of multipotent cells can be omitted from the multipotent cell culture medium prepared to achieve a particular culture purpose.

As used herein, the term "pluripotent cell" refers to a cell capable of differentiating into all three germ cells. Examples of multipotent cells include embryonic stem cells and induced pluripotent stem (iPS) cells. As used herein, “iPS cell” refers to a cell, as described herein, that is genetically substantially identical to its individual differentiated somatic cells of origin and exhibits similar properties to cells with greater pluripotency, such as ES cells. do. These cells can be obtained by reprogramming non-dipotential cells (eg, multipotent cells or somatic cells).

The present invention is directed to a new medium that does not contain factors that are not essential for the specific culture purpose. Examples of culture objectives include, but are not limited to, cell survival, passage, proliferation, multipotency, cloning and iPS cell induction. Specifically, the present invention relates to albumin free medium.

For clarity, "passaging" and "cloning" are different methods. "Passage" refers to a cell division process in which the culture vessel is cultured to a certain density to form aggregates and then transferred to a new culture vessel. These aggregates may comprise any number, typically 100-1,000 cells, and readily initiate proliferation in culture. In contrast, “cloning” refers to initiating clone colonies by growing human ES cell colonies from one individual ES cell. As used herein, “cloning efficiency” means the number of individualized cells forming new cell colonies divided by the number of individualized cells plated in culture. Cloning efficiency is highly dependent on the culture conditions. For example, the cloning efficiency of human ES cells under regulatory and heterogenous-free conditions on MATRIGEL ^® is very low (ie less than about 0.1%), whereas the cloning efficiency of these cells cultured using fibroblast conditions medium Is still low (ie less than about 2%) but high enough to initiate clonal ES cell colonies.

Certain media components currently in use can damage or induce differentiation of cultured cells. For example, β-mercaptoethanol can damage and even kill cultured multipotent cells. Serum medium additives such as bovine serum albumin (BSA) or fetal bovine serum (FCS) can induce differentiation of cultured multipotent cells. In addition, even commercially available serum components may differ greatly in composition even if they are supplied from the same source, thereby introducing unpredictable culture variations. The media described herein are substantially free of damaging, differentiating and non-defining factors present in most conventional multipotent cell culture media. Disclosed media support short-term multipotent cell survival (eg 24 hours, short-term proliferation, eg 4-5 days), maintaining multipotent cells for long periods of culture (eg More than 25 passages in 3 months) and lentiviral and episomal vectors have been used successfully for a variety of culture purposes, such as inducing iPS cells from fetal and adult fibroblasts.

New minimal media tailored specifically for specific cell culture purposes have been developed. Various media components, such as salts, vitamins, glucose sources, minerals and amino acids, were tested alone or in combination to determine the effect of each of them on survival, growth or multiplicity. A novel survival assay was developed and used to identify which components are essential for multipotent cell survival after dissociation. The new medium was tested for its ability to support proliferation and maintain multipotency. These media were also used in cloning assays to determine how each medium affected single cells and their cloning efficiency. The complete list of components for each new medium described herein is shown in Table 1 (light and dark shaded areas indicate the presence of components in the medium, checkered areas indicate interchangeable components, and there is no pattern Region indicates absence of components in the medium).

Various media described herein can be prepared from the basic ingredients. Alternatively, those skilled in the art will understand the beneficial efficiency of using basal medium as starting material to prepare the disclosed novel medium. As used herein, the term “basal medium” means a medium that supports the growth of single cell organisms and cells that do not require special medium additives. Typical basal medium components are known in the art and include salts, amino acids, vitamins and carbon sources (eg, glucose). Other ingredients that do not alter the basic properties of the medium, such as the pH indicator phenol red, but which are otherwise desirable may also be included. For example, Dulbecco's Modified Eagle medium: Nutrient Mixture F-12 (DMEM / F12) is commonly used to prepare suitable growth media for mammalian cell culture. Basic badge. The full list of components of DMEM / F12 is shown in Table 2 below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

In describing an embodiment of the present invention and making the claims, the following terminology will be used in accordance with the definitions set forth below.

As used herein, “about” means within 5% of the stated concentration range or within 5% of the stated time frame.

As used herein, “substantially serum free” means that the medium does not contain serum or serum substitutes or the medium does not substantially comprise serum or serum substitutes. For example, the substantially serum-free medium may comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% serum, At this time the culture capacity of the medium is still observed.

As used herein, the term "defined culture medium" or "defined medium" refers to a medium in which the identity and amount of each medium component is known.

As used herein, "substantially consisting of" means a medium comprising a particular component and optionally including other components that do not substantially affect its basic properties.

As used herein, "effective amount" means an amount sufficient to produce a particular cellular effect in accordance with the present invention.

As used herein, "viability" means a living condition. Viable multipotent cells adhere to the cell plate surface and are not stained with dye propidium iodide without disruption of the cell membrane. Short-term viability relates to the first 24 hours after plating the cells in culture. In general, cells do not proliferate within that time.

As used herein, "short-term growth" refers to cell proliferation for 4-5 days in culture.

As used herein, “extended growth” means growth for five or more passages. Generally, the medium is tested for its ability to support multipotent cell growth for more than 20 passages (about 2-3 months).

As used herein, "long-term growth" means more than 15 passages (about 2 months of culture).

As used herein, "pluripotency" means the ability of a cell to differentiate into cells of all three germ layers.

As used herein, "cloning" refers to the process of initiating cell culture from a starting culture, ideally from a single multipotent cell or from at least very few cells. Culture conditions that allow clone cultures of undifferentiated multipotent cells may be conditions that most demand all of the conditions required for normal multipotent cell culture and proliferation.

As used herein, “iPS cell induction” means reprogramming a cell that is not multipotent to be multipotent.

As used herein, "xeno-free" refers to culture conditions that do not include any cells or other kinds of cell products other than those of cultured cells.

As used herein, "normoxic condition" means a condition comprising about 20% oxygen.

As used herein, "hypoxic condition" means a condition comprising less than about 20% oxygen, for example about 5% oxygen.

The invention will be more fully understood upon consideration of the non-limiting examples described below.

[Example]

Example 1: Multipotent Cell Survival Assays

500 μl of various test media were loaded into each well of a 12 well plate before adding cells. Adherent multipotent cells were dissociated using TrypLE (Invitrogen) for 5 minutes or until the cells were completely detached from the culture plate. TrypLE was neutralized by adding an equal volume of medium to the culture. Cells were counted, washed and resuspended in fresh medium at a concentration of 300,000 to 1,000,000 cells / ml. About 100 μl of this cell solution was added to each well of a 12 well plate and the cells were incubated at 37 ° C. with 5% O ₂ and 10% CO ₂ . 0.4 ml of TrypLE was used to dissociate the cells again at various time points, which were then neutralized with 10% FBS in the same volume of DMEM. Cells were counted by flow cytometry. 5,000 count bright beads were added to each sample as an internal control (about 200 beads were counted for each sample). All experiments were performed in triplicate.

Example 2: Growth Factors for Survival and Short-Term Growth

In addition to being present in the basal medium, TeSR medium contains six growth factors, namely fibroblast growth factor (FGF), transforming growth factor beta (TGF-β), γ-aminobutyric acid (GABA), pipecolic acid and lithium chloride. (LiCl) and insulin (Table 1). A basic nutrient medium (NM) was prepared comprising all TeSR ^™ components except these six growth factors. About 2 × 10 ⁵ H1 ES cells were dissociated and plated on Matrigel. Survival index was measured after 24 hours. NM alone could not support cell survival after dissociation. Addition of insulin to NM induced cell survival similar to that observed in TeSR ^™ , but did not support cell growth (FIG. 1A). Addition of both insulin (20 μl / ml) and FGF2 (100 ng / ml) supported cell survival and induced cell growth after an additional 96 hours, which was similar to that observed with TeSR ^™ medium ( 1b). Thus, NM supplemented with FGF and insulin supports human ES cell culture. Twelve different basal nutrient media supplemented as described above could support cell survival and growth (FIG. 1E).

Example 3: L-ascorbic acid supports short term growth.

NM includes 11 nutrients, DMEM / F12, trace element B, trace element C, L-ascorbic acid, thiamine, selenium, L-glutamine, BSA, BME, sodium bicarbonate (NaHCO ₃ ) and transferrin ( Table 1). DMEM / F12 acts as a basal medium and NaHCO ₃ is used to adjust the pH. Each factor was added separately to DMEM / F12, NaHCO ₃ , insulin and FGF to confirm if other nutritional components are essential when insulin and FGF are present. None of the nutritional factors was essential for survival after passage, but L-ascorbic acid (64 mg / L) was required for cell proliferation after passage (FIG. 1C). L-ascorbic acid, known as vitamin C, is a major antioxidant and cofactor of several enzymes. Hydroxyproline may partially replace L-ascorbic acid. Human ES cells plated in DMEM / F12, NaHCO ₃ , L-ascorbic acid, insulin and FGF (regulation factor 5, “DF5”, Table 1) have similar morphology to human ES cells plated with TeSR. Maintained.

Example 4: Medium Components for Long-Term Passage

DF5 supported cell growth for only one passage. After two passages, the cells did not adhere well and eventually died (FIG. 1C). Cells could be passaged between NM + insulin + FGF (data not shown) and DF12 (FIG. 1D, Table 1), indicating that one or more factors present in NM + insulin + FGF and DF12 are important for long-term passage. Suggests that. Each nutrient factor present in NM was individually added to DF5 to determine its ability to support cell proliferation after multiple passages. The addition of selenium alone was sufficient to support cell proliferation through multiple passages (FIG. 1D, DF5 + Selenium, “DF5S,” Table 1).

DF5S was used to propagate H1 cells. Cells cultured in DF5S tended to differentiate better than cells cultured in TeSR ^™ . However, H1 cells were able to grow for several weeks (more than 15 passages), during which time cells maintained human ES cell morphology and high levels of OCT4 expression (FIG. 1E, FIG. 1F). H1 cells cultured in DF5S with NODAL (100 ng / ml) or TGF-β (2 ng / ml) added significantly higher levels of NANOG mRNA compared to H1 cells cultured in DF5S . DF5S + NODAL also supported the multipotency of the two tested human iPS cell lines, as confirmed by high levels of expression of the multipotency marker OCT4. All cells (hES cells and iPS cells) cultured in DF5S with NODAL or TGF-β added maintained their normal karyotype after long passage.

Example 5 Hypoxia Improves Cell Growth and Cloning

H1 cells grow better in DF5S compared to cells cultured in TeSR ^™ (FIGS. 1C and 2A). To optimize multipotent cell growth conditions, cells were cultured in DF5S with various osmotic concentrations, pH, oxygen levels and CO ₂ levels. To increase assay sensitivity, only 5,000 cells were seeded into each well and analyzed for survival (24 hours) and proliferation (124 hours). Maximum improvement was observed when the O ₂ and CO ₂ levels were varied. Normal culture conditions use about 15% oxygen and 5% CO ₂ (O15C5). Higher levels of CO ₂ often slightly improved survival after 24 hours. Lower oxygen levels increased cell growth in both DF5S and TeSR ^™ . 10% CO ₂ and 15% O ₂ (O 15 C 10) and 10% CO ₂ and 5% oxygen (O 5 C 10) increased cell survival (FIG. 2A). Cells did not thrive at higher O ₂ levels (O ₁₅ C 5 and O ₁₅ C 10), while proliferating at lower oxygen levels (O 5 C 10) (FIG. 2A). Cells in DF5S grew faster than cells cultured in TeSR ^™ and grew fastest at 5% O ₂ and 10% CO ₂ (FIG. 2A). Further reduction of oxygen levels to 2% resulted in a decrease in cell growth compared to 5% O ₂ .

To measure cloning efficiency at various oxygen and CO ₂ concentrations, 500 cells were seeded into each well. Even at low oxygen levels, the cloning efficiency was too low (<2%) to confirm the effects of various conditions upon cloning. HA100, a ROCK inhibitor known to increase cloning efficiency, was used to increase cloning efficiency to test oxygen and CO ₂ concentrations. Conditional medium (CM), known to be the best medium for cloning, was used as a control. The addition of HA100 markedly improved the cloning efficiency in CM at both O5C10 and O15C5, and the cloning efficiency was higher at O5C10 than O15C5 (FIG. 2B). The cloning efficiency of cells in DF5S was comparable to the cloning efficiency of cells in CM under both conditions (FIG. 2B).

Due to the positive effect of hypoxic conditions on cell survival, some of the subsequent examples utilize hypoxic conditions when the cells are kept at low density. However, when the cells were not incubated at low cell density, the experiment was performed at both normal oxygen and hypoxic conditions (FIG. 2B).

Example 6: Improved iPS Cell Cloning Efficiency

To see how DF5S affects cloning efficiency, two iPS cell lines were cultured in DF5S and plated at cloning density (about 500 cells per 12 well plate well) in the presence of HA100. The cloning efficiency of iPS cells cultured in DF5S was lower than the cloning efficiency of iPS cells cultured in TeSR ^™ or CM (FIG. 2C), which indicates that factors that enhance cloning efficiency are present in TeSR ^™ medium, but not in DF5S. Suggests. To confirm that factor, individual TeSR ^™ components were added individually to DF5S to test for impact on cloning efficiency. Adding holotransferrin to DF5S (DF5SFe) led to similar cloning efficiency as using TeSR ^™ (FIG. 2D). Transferrin also led to a significant improvement in the cloning efficiency of H1 cells in DF5S medium.

ROCK inhibitors HA100 and Y27632, and blevistatin, in DMEM / F12 ("E8") supplemented with insulin, transferrin, selenium, L-ascorbic acid, FGF, and TGF-β (or NODAL) were effective for cloning H1 cells. Was increased (FIG. 2E), which was further increased by the addition of transferrin and incubation under hypoxic conditions (FIG. 2F). The cells maintained a normal karyotype after more than 25 passages.

Example 7 NODAL and TGF-β Support Long-term Maintenance of H1 and iPS Cell Multidifferentiation in Albumin Medium

As described in Example 3, human pluripotent cells such as H1, H9 and iPS cells were grown in DF5S and passaged more than 15 times, but tended to differentiate, requiring special attention to maintain pluripotency in DF5S It was. Since multipotency in TeSR ^™ can be more easily maintained, growth factors present in TeSR ^™ were added separately to the DF5SFe used to culture H1 cells, and the cells identified factors that support long-term multidifferentiation. To be precultured in DF5S without differentiation. Approximately one day after reaching confluency, cells were passaged to promote cell differentiation and Oct4 expression measured by flow cytometry was used as an indicator of multidifferentiation.

Human pluripotent cells cultured in DF5SFe grew long and lined up along each other, becoming a "spindle" -like shape just before differentiation. This phenotype is often observed at the onset of neural differentiation, which is generally inhibited by the TGF-β BMP pathway. Thus, recombinant proteins of the TGF-β pathway were tested for their ability to support long term multiplicity. DF5SFe (“E8 (NODAL)”) supplemented with NODAL used at TeSR ^™ concentrations maintained high levels of Oct4 expression. DF5SFe ("E8 (TGF-β)") supplemented with TGF-β used at TeSR ^TM concentration (0.6 ng / ml) supported low levels of Oct4 expression, but at higher concentrations (1 ng / ml) High levels of Oct4 expression could be maintained.

Human ES cell lines such as H1 and H9 have a culture history including exposure to various complex culture components such as FBS, feeder cells and knockout serum substitutes. Exposure to these components may lead to dependence on these components, and consequently alter the cellular response to simplified media. The culture history may be less important for iPS cells derived from reprogrammed somatic cells because the induction conditions are less complex. Therefore, various factors were tested using two original lentiviral iPS cell lines (Yu, et al. , Science 318: 1917 (2007)) cultured in DF5SFe. Cells were transferred directly from MEF plates to DF5SFe medium for one passage and then passaged to various growth factor conditions. Addition of TGF-β (2 ng / ml) or NODAL (100 ng / ml) to DF5SFe ("E8 (TGF-β)" and "E8 (NODAL), respectively)) supported long-term multipotency of iPS cells. Multipotent surface markers SSEA4, SSEA3, Tra-1-60 and Tra-1-81 were also expressed. Cells with normal karyotype remained steady for more than 20 passages. The cells were able to form teratomas 5-7 weeks after injection into severe combined immunodeficiency (SCID) mice.

E8 (TGF-β) and E8 (NODAL) are all multipotent cells tested for more than 25 passages (approximately 3 months), that is, multiples of two human ES cell lines (H1 and H9) and five iPSC cell lines. Mars was supported and there were no signs of differentiation (FIG. 3). H1 ES cells cultured in E8 medium have a similar gene expression profile compared to H1 ES cells cultured in TeSR ^™ (FIG. 3B).

Example 8: Induction of iPS Cells in Albumin Medium

Reprogramming protocols available include FBS within the first few days after viral transduction or electroporation, before exchanging cells for UM100 (US Pat. No. 7,439,064, which is incorporated herein in its entirety herein) or CM. Incubating the cells in the middle. The simplified media described in the previous examples were tested for their ability to support reprogramming. ES induced somatic cells could be efficiently reprogrammed in DF5S medium using lentiviral or episomal vectors with or without the first two days of culture in FBS containing medium. However, DF5S did not support reprogramming of primary foreskin cells when using Nanog, Oct4, Sox2 and Lin28. DF5SFe supported reprogramming of foreskin cells and adult cells on Matrigel or MEF when using an improved lentivirus (Ebert et al. , Nature 457 (7227): 277-280 (2009), which is entirely Included herein as described herein), wherein the cells were initially incubated in FBS containing medium. DF5SFe was as effective as CM in supporting reprogramming, but initial exposure to FBS appeared to be important for reprogramming.

Foreskin cells grow much slower in DF5SFe than in FBS medium. In order to identify growth factors that may help primary foreskin cell growth, individual growth factors contained in FBS were tested. The FGF growth factor family includes several members, one or more of which are commonly used for fibroblast culture. DF5SFe contains 100 ng / ml zebrafish recombinant FGF2. Each FGF family member was tested for its ability to support foreskin cell growth. Foreskin cells were aliquoted into wells of the culture plate and incubated for 24 hours in DF5SFe without FGF. Each type of FGF was added at 100 ng / ml for 96 hours. FGF1, zFGF2, FGF4, FGF6 and FGF9 most effectively supported foreskin cell growth, but neither FBS containing medium nor cell growth (FIG. 4A). Several known fibroblast growth promoters were tested to see if non-FGF family member growth factors could promote foreskin cell growth similar to that observed in FBS. Hydrocortisone (FIG. 4B), its derivatives and dexamethasone added to DF5SFe to replace FBS markedly improved cell growth. DF5SFe + hydrocortisone (“DF5SFeC”) also improved iPS cell cloning efficiency.

To ascertain whether media based on DF5S can be used for induction of virus-free iPS cells, as described herein in its entirety, see Yu et al. , Science 324: 797 (2009), reprogrammed foreskin cells using hypoxic episomal vectors under hypoxic conditions (O5C10). Plasmid combination # 4 (pEP4EP2SCK2MEN2L and pEP4EO2SET2K, Table 3), # 6 (pEP4EO2SEN2L, pEP4EO2SET2K and pEP4EO2SEM2K, Table 3) and # 19 (pEP4EO2SEN2K, pEP4EO2SET2K and pCEP4-M2L, Table 3), 10 ⁶ after the second passage was used Two clones were isolated from dog cells.

Plasmid combinations # 6 and # 19 were used for reprogramming. To enhance plasmid entry into the nucleus, ENBA mRNA was electroporated with plasmid DNA. About 1 million cells were transferred in two 6 well plates of DF5SFeC and incubated for 5 days. Thereafter, the medium was replaced with DF5SFe and further incubated for 18-25 days. Cells from some wells were passaged secondly using a ratio of 1: 6 at different time points. Plasmid Combination # 19 produced more colonies than Plasmid Combination # 6, but most of them did not resemble typical human ES cell morphology. After about 25 days, human ES cell-like clones appeared on the primary plates for the two plasmid combinations, with an estimated number of 24 reprogramming cells per million foreskin cells when using plasmid combination # 19, plasmid combination # Using 6, the estimated number of reprogramming cells per million foreskin cells was eight. The number of human ES cell-like colonies increased significantly after the second passage plate and the estimated number per million foreskin cells for each plasmid combination was less than 500. The increase in iPS cell colonies on the secondary passage plate is probably due to the division of iPS cells on the primary plate. In some cases, the primary plate did not have any colonies resembling a typical human ES cell morphology, but many iPS cells appeared after the secondary passage, suggesting that some iPS cells could not be identified, presumably they were somatic It is probably because it is mixed with.

Cells of iPS cell colonies derived in DF5SFe began to differentiate after only two passages. Six iPS cell colonies were picked from primary plates and transferred directly to Nodal containing DF5SFeN (E8 (NODAL)). These cells were maintained for more than 15 passages in E8 (Nodal), where they maintained an ES cell-like morphology similar to that observed when using TeSR ^™ . The cells had a normal karyotype and expressed Oct4 and SSEA4 (FIG. 4C) and formed teratomas in SCID mice 5-7 weeks after injection.

In addition, foreskin fibroblasts were reprogrammed in E8 medium. Total gene expression of iPS cells induced in E8 medium was similar to iPS cells (FIGS. 4D and 4F) induced on H1 cells (FIGS. 4D and 4E) or feeder cells. Multipotent markers were highly expressed in both ES cells and iPS cells, but did not express fibroblast specific marker genes (FIG. 4D). In addition, iPS cells can be induced in E8 medium using a variety of strategies, for example using lentiviral or episomal vectors.

Example 9: Induction of iPS Cells from Patient Cell Lines in Albumin Medium

To confirm that cells from adult donors can be reprogrammed using virus-free episomal vectors in a simplified medium, 2 million patient cell lines OAT or PRPT8 cells were transfected with plasmid combination # 4 or # 6 with EBNA mRNA. Perforated and transferred onto two 10 cm plates. To maximize reprogramming, FBS containing media was used for the first 6 days. To maintain constant adult cell maintenance conditions, cells were maintained at O15C5. Thereafter, the medium was replaced with DF5SFe and cultured further for 14 to 21 days. Cells from one plate were passaged at a ratio of 1: 2 at different time points. Plasmid combination # 6 produced more colonies (about 5 per million cells) than # 4, but most of the cells did not resemble typical human ES cell morphology. After about 22 days, human ES cell-like colonies appeared on the primary plate for plasmid combination # 4. More human ES cell-like colonies appeared on the secondary passage plate when plasmid combination # 6 was used, with about 40 colony estimates per million cells. IPS cells were not produced when plasmid combination # 4 was used. IPS cell colonies appeared in the middle of other densely clustered cells on the primary plate, which could not proliferate beyond their boundaries. However, colonies on the secondary plate were grown to large sizes suitable for colony isolation. Colonies were picked and transferred directly to TeSR ^™ , and 32 picking colonies survived to show ES cell morphology. Genetic analysis confirmed that these colonies were derived from OAT cell lines and showed normal karyotypes.

To improve adult cell reprogramming efficiency, TGF-β was added to the reprogramming medium. iPS clones did not increase significantly, but total colony counts increased significantly. When TGF-β was removed from the medium upon hydrocortisone removal, the number of iPS cell colonies increased significantly, indicating that TGF-β supports reprogramming within the first few days of this process.

Many apparent non-iPS colonies can produce iPS clones after the second pass, suggesting that iPS cell induction can be inhibited by surrounding cells. Several reagents were tested to test their ability to overcome this effect. Butyrate improved reprogramming efficiency. An about 10-fold increase in reprogramming efficiency of foreskin cells was observed when both TGF-β and butyrate were added to the medium (FIG. 5B). TGF-β appeared to have a positive effect during the early stages of reprogramming, but butyrate played a positive role in later stages. TGF-β addition resulted in an increase in the number of colonies during reprogramming, but the number of original iPS cell colonies was kept low. Butyrate did not increase the number of colonies, but significantly improved the ratio of native iPS cells to non-iPS cell colonies (FIG. 5C).

The use of TGF-β and butyrate enabled the successful reprogramming of somatic cells from adult individuals under fully defined conditions using an episomal vector system. iPS cells were isolated from three independent adult somatic cell lines (OAT, GRC M1-29 and PRPF8-2) with the efficiency of 1 to 100 of 1 × 10 ⁶ PRPF8-2 cells and 1 of 100,000 cells (GRC 1-29). Derived from.

Example 10 Induction of iPS Cells from Adult Subjects in Fully Defined Conditions

Biopsies are taken from the skin of an adult adult donor and washed with Hank's Buffered Salt Solution (HBSS) containing antibiotics and antifungal agents and 4 ° C. in 2 ml of 0.25% trypsin / EDTA (Table 4) or TrypLE Select. Incubated overnight at. Samples were rinsed three times and trypsin inhibitor was used after the second rinse (Table 4). Sterile tweezers were used to separate the dermis from the epidermis. The dermis was cut into small pieces and incubated for 3 hours in 0.75 ml of enzyme solution (Table 4) containing the desired enzyme at room temperature (12 well or 24 well plates). After about 35 minutes, the tissue structure began to degrade. An equal volume of medium containing 10 μl / ml polyvinylpyrrolidone (PVP) was added and the tissue was physically disassembled with about 10 pipetting up / down. Samples were centrifuged at 400 g for 10 minutes at room temperature and washed twice with fresh medium / PVP. The supernatant was discarded and the pellet was resuspended in 3 ml of complete medium and 1 ml of cell suspension was transferred to wells of 6 well plates coated with 3 μg Vitronectin per well. Plates were incubated at 37 ° C. with 5% CO ₂ and the medium changed daily. Fibroblasts adhered to the plate, but non-adherent cells and debris were removed when the medium was changed.

After 20 days, reprogramming plasmids were introduced into fibroblasts using electroporation. Within the next 25 days, multiple iPS colonies appeared and picked for further analysis. The reprogramming efficiency was about 10 per million electroporated fibroblasts without secondary passage. IPS cells were further passaged to isolate cell lines without vectors.

Example 11: Inducing iPS Cells from Adult Subjects in Albumin Medium Without Secondary Passage

Adult fibroblasts were reprogrammed in E8 (DMEM / F12 supplemented with insulin, transferrin, selenium, L-ascorbic acid, FGF2 and TGF-β (or NODAL)) according to the general protocol illustrated in FIG. 6A. Reprogrammed iPS cell lines maintained in E8 for more than 20 passages consistently expressed multipotent markers OCT4 and SSEA4 (FIG. 6B).

E8 media significantly improved reprogramming efficiency compared to reprogramming efficiency when using mouse fibroblast feeder cells (MEF) (FIG. 7A) or TeSR ^™ (FIG. 7B). Butyrate (100 μM) further improved reprogramming efficiency in the presence of TGF-β (E8) or in the absence of TGF-β (TGF-β free E8, ie DF5SFe) (FIG. 7C).

Example 12 Cryopreservation of Multipotent Stem Cells in Albumin-Free Medium

As substantially described above, multipotent cells were cultured in E8 medium in 6 well plates. Culture medium was aspirated from each well and cells were washed twice with 1.0 mL of EDTA / PBS (0.5 mM EDTA in PBS, osmotic concentration 340). Cells were then incubated at 37 ° C. in EDTA / PBS for 5 minutes. PBS / EDTA was removed and cells were quickly rinsed with 1 ml of E8 medium. Cells are then resuspended in equal volumes of 20% dimethyl sulfoxide (DMSO) and E8 medium (final concentration: 10% DMSO in E8 medium), aliquoted into cryogenic vials and -80 using CRYOBOX ^™ . Frozen at ° C. Thereafter, the cells were transferred to a liquid nitrogen tank.

The present invention has been described with reference to embodiments which are presently considered to be the most practical and preferred embodiments. However, the present invention has been presented by way of example only, and is not intended to be limiting to the disclosed embodiments. Accordingly, those skilled in the art will understand that all modifications and alternative constructions within the spirit and scope of the invention disclosed in the appended claims are intended to be included.

Claims (29)

Albumin free media supporting the proliferation of multipotent stem cells. The medium of claim 1 comprising water, salts, amino acids, vitamins, carbon sources, insulin, FGF, selenium, transferrin, and one of TGF-β and NODAL in an amount sufficient to support multipotent stem cell proliferation, respectively. As a method of culturing multipotent stem cells,
Positioning multipotent stem cells on the matrix; And
Contacting the cells with an albumin-free medium that supports multipotent stem cell proliferation
&Lt; / RTI &gt; The method of claim 3, wherein the medium comprises water, salts, amino acids, vitamins, carbon sources, insulin, FGF, selenium, transferrin, and one of TGF-β and NODAL, respectively, in an amount sufficient to support multipotent stem cell proliferation. How to do. The method of claim 3, wherein the matrix comprises laminin. The method of claim 3, wherein the matrix comprises Vitronectin. The method of claim 3, wherein said cells are contacted with said medium under hypoxic conditions. The method of claim 3, wherein said multipotent stem cells are embryonic stem cells. The method of claim 3, wherein said multipotent stem cells are induced pluripotent stem cells. The method of claim 3, wherein said multipotent stem cells are human cells. A method of cloning pluripotent stem cells, the method comprising plating the pluripotent stem cells at a cloning density in albumin-free medium that supports pluripotent stem cell cloning. The method of claim 11, wherein the medium comprises water, salts, amino acids, vitamins, carbon sources, insulin, FGF, selenium, transferrin, and one of TGF-β and NODAL, respectively, in an amount sufficient to support multipotent stem cell cloning. How to do. The method of claim 11, wherein the medium further comprises a ROCK inhibitor. The method of claim 13, wherein the ROCK inhibitor is selected from the group consisting of HA100 and Y27632. The method of claim 11, wherein the medium further comprises blevistatin. A method of cryopreserving pluripotent stem cells, the method comprising freezing the pluripotent stem cells in albumin free medium. The method of claim 16, wherein the medium comprises water, salts, amino acids, vitamins, carbon sources, insulin, FGF, selenium, transferrin, and one of TGF-β and NODAL in an amount sufficient to support multipotent stem cell proliferation, respectively. How to do. A method of inducing induced pluripotent stem (iPS) cells under defined conditions, the method comprising reprogramming somatic cells in albumin-free media supporting somatic reprogramming to induce iPS cells . The method of claim 18, wherein the medium comprises water, salts, amino acids, vitamins, carbon sources, insulin, FGF, selenium, transferrin, and one of TGF-β and NODAL, respectively, in an amount sufficient to support iPS cell induction. How to be. The method of claim 18, wherein the reprogramming step comprises contacting the cells with TGF-β for 5-10 days. 21. The method of claim 20,
Removing TGF-β after 5-10 days; And
Contacting the cells with a medium consisting substantially of water, salts, amino acids, vitamins, carbon sources, insulin, FGF, selenium, and transferrin
&Lt; / RTI &gt; The method of claim 18, wherein said reprogramming step comprises contacting said cells with hydrocortisone. The method of claim 18, wherein said reprogramming step comprises contacting said cells with butyrate. The method of claim 18, wherein the iPS cells are induced under xeno-free conditions. 19. The method of claim 18, wherein said somatic cells are adult somatic cells. 19. The method of claim 18, wherein said reprogramming step comprises using a viral vector. 19. The method of claim 18, wherein the reprogramming step comprises using an episomal vector. Substantially made up of one of water, salt, amino acid, vitamin, carbon source, insulin, FGF, selenium, transferrin, and TGF-β and NODAL, in an amount sufficient to support a long-term multipotent stem cell culture, respectively. Albumin-free growth medium supporting long-term culture of multipotent stem cells. As a method of culturing multipotent stem cells,
Positioning the multipotent stem cells on the matrix; And
The cells are albumin-free, consisting essentially of one of water, salts, amino acids, vitamins, carbon sources, insulin, FGF, selenium, transferrin, and TGF-β and NODAL, in an amount sufficient to support long-term multipotent stem cell culture, respectively. Contact with the medium
&Lt; / RTI &gt;

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